Platelet targeted treatment

ABSTRACT

The present disclosure relates to compositions and methods for targeting expression of exogenous genes to platelets. In particular, the present disclosure relates to treatment of hemophilia and other diseases and conditions by targeting expression of exogenous agents (e.g., clotting factors) to platelets.

This application is a continuation of U.S. application Ser. No.15/689,875, filed Aug. 29, 2017, now U.S. Pat. No. 10,294,291, IssuedMay 21, 2019, which is a divisional of U.S. application Ser. No.14/437,457, filed Apr. 21, 2015, now U.S. Pat. No. 9,982,034, Issued May29, 2018, which is a national phase application under 35 U.S.C. § 371 ofPCT International Application No. PCT/US2013/066651 filed Oct. 24, 2013,which claims priority to U.S. Provisional Application No. 61/717,951filed Oct. 24, 2012, each of which are herein incorporated by referencein its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. R01HL068138 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present disclosure relates to compositions and methods for targetingexpression of exogenous genes to platelets. In particular, the presentdisclosure relates to treatment of hemophilia and other diseases andconditions by targeting expression of exogenous agents (e.g., clottingfactors) to platelets.

BACKGROUND OF THE INVENTION

Hemophilia is a common bleeding disorder (occurring in approximately1:10,000 males) in which causes severe internal bleeding that oftenleads to death because the patient's blood doesn't clot normally.Hemophilia usually is inherited with patients displaying severeuncontrollable bleeding events beginning at birth and re-occurringthroughout the individual's life. Although there are several types ofclotting factors that work together with platelets to help the bloodcoagulate, people with hemophilia usually have quantitative orqualitative defects in the proteins that encode coagulation factor VIII(hemophilia A) or factor IX (hemophilia B) that prevent normalhemostasis.

Hematopoietic stem cells differentiate in the bone marrow to formmegakaryocytes that mature and break-up into several thousand smallfragments known as platelets, which in normal conditions circulatequietly (not interacting with the other blood cells or the vessel wall)in the blood stream for approximately 10 days with the main job tobecome activated, change shape and stick to damaged blood vessel torepair the injury. When blood vessels are injured, clotting factors helpplatelets stick together to plug cuts and close breaks on the vessels tostop bleeding.

People with hemophilia A are missing or have low levels of clottingfactor VIII. About 9 out of 10 people who have hemophilia have type A.People with hemophilia B are missing or have low levels of clottingfactor IX. Both clotting factors are normally synthesized in the liveralthough there are reports that other cell types can be induced tosynthesize fully functional forms of recombinant FVIII and FIX proteins.

Hemophilia can be mild, moderate, or severe, depending on how muchnormal functional clotting factor is present in the blood. About 7 outof 10 people who have hemophilia A have the severe form of the disorder.

Hemophilia usually occurs in males because Factors VIII and IX arelocated on the X chromosome (although with rare exceptions females whoinherit a defective X chromosome each from an affected father and motherwho is a carrier for the disease). About 1 in 10,000 individuals areborn with hemophilia each year all over the world.

The main treatment for hemophilia is protein replacement therapy.Concentrates of clotting factor VIII (for hemophilia A) or clottingfactor IX (for hemophilia B) can be isolated from pools of donor bloodor recombinant protein that has been prepared from tissue culture celllines transformed with the normal genes encoding FVIII or FIX that areslowly dripped or injected into a vein at the onset of a seriousbleeding event. These infusions help replace the clotting factor that'smissing or low.

Complications of replacement therapy include developing antibodiesresponse to the normal therapeutic protein that is foreign to thepatient's immune system (known as inhibitor formation), which ultimatelyleads to inactivation or destruction of the clotting factor anduncontrolled bleeding in about 30% of patients, developing viralinfections from human clotting factors (from blood contaminated with HIVor Hepatitis from infected blood donors especially in third worldcountries), very expensive costs of the replacement protein which has avery short half-life (days) which requires frequent re-administration tosubside a severe vascular injury and damage to joints, muscles, or otherparts of the body resulting from delays in treatment.

Thus, new treatments for hemophilia that overcome these complicationsare needed.

SUMMARY OF THE INVENTION

The present disclosure relates to compositions and methods for targetingexpression of exogenous genes to platelets. In particular, the presentdisclosure relates to treatment of hemophilia and other diseases andconditions by targeting expression of exogenous agents (e.g., clottingfactors) to platelets. In some embodiments, the present disclosurerelates to compositions and clinically relevant methods forhematopoietic stem cell gene therapy where targeting expression ofexogenous genes within bone marrow megakaryocytes leads to expressionand/or storage of recombinant therapeutic proteins within humanplatelets.

For example, in some embodiments, the present invention provides acomposition comprising an expression vector comprising a) an expressioncassette comprising a fragment of the integrin αIIb gene (ITGA2B)promoter; and an exogenous gene of interest operably linked to theexpression cassette. In some embodiments, the expression cassettecomprises, consists essentially of, or consists of a nucleic acidsequence selected from, for example, SEQ ID NOs: 21, 22, or 23, orsequences larger or smaller than SEQ ID NOs:21, 22, 23, or 25 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 75, 100, 150, 200, 300,400, 500, 600 or more nucleotides larger or smaller than SEQ ID NOs: 21,22, 23, and 25) or fragments thereof. In some embodiments, theexpression vector further comprises targeting factor (e.g., a fragmentof the human Von Willebrand Factor propeptide (VWFpp) operably linked toa D2 domain) (e.g., as described by SEQ ID NO:24 or sequences 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 75, 100, 150, 200, 300, 400,500, 600 or more nucleotides larger or smaller than SEQ ID NO: 24 orfragment of SEQ ID NO:24). The present invention is not limited to aparticular exogenous gene. Examples include, but are not limited to, ahuman FVIII or FIX gene. In some embodiments, the vector is aself-inactivating vector, for example, a retroviral vector (e.g., alentiviral vector).

Further embodiments provide a hematopoietic stem cell comprising theexpression vectors described herein (e.g., an ex vivo stem cell).

In yet other embodiments, the present invention provides the use of suchstem cells to treat diseases and conditions (e.g., hemophilia) in ananimal (e.g., a human).

The present invention also provides a method, comprising: contacting ahematopoietic stem cell with a vector as described herein to generate amodified stem cell under conditions such that a gene (e.g., Factor VIIIgene) is expressed in the modified stem cell. In some embodiments, themethod further comprises the step of transferring said modified stemcell into an animal (e.g., human). In some embodiments, the animal hasbeen diagnosed with hemophilia and the transferring treats or preventsexcessive bleeding in the animal. In some embodiments, the contactingoccurs ex vivo. In some embodiments, the stem cells are mobilized fromthe animal (e.g., by administration of cytokines, mobilization intoperipheral blood, contacting apheresis collection, immune magnetic beadisolation). In some embodiments, the animal expresses said Factor VIIIin platelets. In some embodiments, the method is repeated (e.g., atregular intervals or when needed).

Additional embodiments are described herein.

DESCRIPTION OF THE FIGURES

FIG. 1 shows platelet-targeted lentiviral vector design. (A) ITGA2B genepromoter fragments direct megakaryocyte-specific expression inluciferase. (B) −889ITGA2B-BDDFVIII-WPTS lentiviral vector diagram. (C)−673ITGA2B-VWFSPD2-BDDFVIII-WPTS lentiviral vector diagram.

FIG. 2 shows synthesis and trafficking of BDDFVIII into canine plateletα-granules (A) confocal microscopy showing co-localization of BDDFVIIIand Fg within platelets. (B) electron microscopy localized humanBDDFVIII directly in α-granules.

FIG. 3 shows quantitative analysis of platelet FVIII.

FIG. 4 shows activated platelets induced to secrete FVIII:C.

FIG. 5 shows PCR analysis for detection and localization of lentiviralvector within canine genome. (A) long-term detection ofBDDFVIII-lentiviral vector within leukocyte genomic DNA. (B) linearamplification-mediated (LAM)-PCR to localize lentiviral vector withincanine genome.

FIG. 6 shows correction of the canine Hemophilia A phenotype withplatelet BDDFVIII.

FIG. 7 shows structural regions of the ITGA2B gene promoter and VWFspD2.Arrows show serious bleeding events before and after platelet targetedtreatment. Dogs 142 and M64 show complete correction of hemostasis

FIG. 8 shows an exemplary lentiviral gene therapy vector of embodimentsof the present disclosure.

FIG. 9 shows the sequence (SEQ ID NO:25) of the vector of FIG. 8.

FIG. 10 shows a diagram and nucleotide sequence for integrin αIIbpromoter fragments used in recombinant lentivirus gene transferconstructs. (A) Nucleotide sequence for -Sal I Bgl II −1218 to +30 ofhuman αIIb-gene promoter Nco I used for Megakaryocyte-SpecificLuciferase reporter studies (SEQ ID NO:21). (B) Nucleotide sequence for-Sal I Bgl II −889 to +30 of human αIIb-gene promoter Nco I used forMegakaryocyte-Specific Luciferase reporter studies (SEQ ID NO:22). (C)Nucleotide sequence for -Sal I Bgl II −673 to +30 of human αIIb-genepromoter Nco I used for Megakaryocyte-Specific Luciferase reporterstudies (SEQ ID NO:23). Numbering is based on Prandini, et al (BiochemBiophys Res Commun 156(1) 595-601, 1988).

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the term “gene transfer system” refers to any means ofdelivering a composition comprising a nucleic acid sequence to a cell ortissue. For example, gene transfer systems include, but are not limitedto, vectors (e.g., retroviral, adenoviral, adeno-associated viral, humanartificial chromosomes, and other nucleic acid-based delivery systems),microinjection of naked nucleic acid, polymer-based delivery systems(e.g., liposome-based and metallic particle-based systems), biolisticinjection, and the like. As used herein, the term “viral gene transfersystem” refers to gene transfer systems comprising viral elements (e.g.,intact viruses, modified viruses and viral components such as nucleicacids or proteins) to facilitate delivery of the sample to a desiredcell or tissue. As used herein, the term “adenovirus gene transfersystem” refers to gene transfer systems comprising intact or alteredviruses belonging to the family Adenoviridae.

As used herein, the term “nucleic acid molecule” refers to any nucleicacid containing molecule, including but not limited to, DNA or RNA. Theterm encompasses sequences that include any of the known base analogs ofDNA and RNA including, but not limited to, 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxy-aminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of apolypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction,immunogenicity, etc.) of the full-length or fragment are retained. Theterm also encompasses the coding region of a structural gene and thesequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb or more on either end such that thegene corresponds to the length of the full-length mRNA. Sequenceslocated 5′ of the coding region and present on the mRNA are referred toas 5′ non-translated sequences. Sequences located 3′ or downstream ofthe coding region and present on the mRNA are referred to as 3′non-translated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns maycontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide.

As used herein, the term “heterologous gene” refers to a gene that isnot in its natural environment. For example, a heterologous geneincludes a gene from one species introduced into another species. Aheterologous gene also includes a gene native to an organism that hasbeen altered in some way (e.g., mutated, added in multiple copies,linked to non-native regulatory sequences, etc). Heterologous genes aredistinguished from endogenous genes in that the heterologous genesequences are typically joined to DNA sequences that are not foundnaturally associated with the gene sequences in the chromosome or areassociated with portions of the chromosome not found in nature (e.g.,genes expressed in loci where the gene is not normally expressed).

Promoter/enhancer,” as used herein, denotes a segment of DNA whichcontains sequences capable of providing both promoter and enhancerfunctions (i.e., the functions provided by a promoter element and anenhancer element, see above for a discussion of these functions). Forexample, the long terminal repeats of retroviruses contain both promoterand enhancer functions. The enhancer/promoter may be “endogenous” or“exogenous” or “heterologous.” An “endogenous” enhancer/promoter is onethat is naturally linked with a given gene in the genome. An “exogenous”or “heterologous” enhancer/promoter is one that is placed injuxtaposition to a gene by means of genetic manipulation (i.e.,molecular biological techniques such as cloning and recombination) suchthat transcription of that gene is directed by the linkedenhancer/promoter. Regulatory elements may be tissue specific or cellspecific. The term “tissue specific” as it applies to a regulatoryelement refers to a regulatory element that is capable of directingselective expression of a nucleotide sequence of interest to a specifictype of tissue (e.g., mammary gland) in the relative absence ofexpression of the same nucleotide sequence(s) of interest in a differenttype of tissue (e.g., liver). Tissue specificity of a regulatory elementmay be evaluated by, for example, operably linking a reporter gene to apromoter sequence (which is not tissue-specific) and to the regulatoryelement to generate a reporter construct, introducing the reporterconstruct into the genome of an animal such that the reporter constructis integrated into every tissue of the resulting transgenic animal, anddetecting the expression of the reporter gene (e.g., detecting mRNA,protein, or the activity of a protein encoded by the reporter gene) indifferent tissues of the transgenic animal. The detection of a greaterlevel of expression of the reporter gene in one or more tissues relativeto the level of expression of the reporter gene in other tissues showsthat the regulatory element is “specific” for the tissues in whichgreater levels of expression are detected. Thus, the term“tissue-specific” (e.g., liver-specific) as used herein is a relativeterm that does not require absolute specificity of expression. In otherwords, the term “tissue-specific” does not require that one tissue haveextremely high levels of expression and another tissue have noexpression. It is sufficient that expression is greater in one tissuethan another. By contrast, “strict” or “absolute” tissue-specificexpression is meant to indicate expression in a single tissue type(e.g., liver) with no detectable expression in other tissues.

As used herein the term “portion” or “fragment” when in reference to anucleotide sequence (as in “a portion or fragment of a given nucleotidesequence”) refers to fragments of that sequence. The fragments may rangein size from four nucleotides to the entire nucleotide sequence minusone nucleotide (10 nucleotides, 20, 30, 40, 50, 100, 200, etc.). In someembodiments, fragments comprise a nucleotide sequence (e.g., promoter)that are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 30, 40, 50, 100, 150, or 200 nucleotides less than the sequenceor subsets thereof (e.g., 31, 32, 33, 34, 35, 35, 36, 37, 38, 39nucleotides shorter and the like).

As used herein, the term “oligonucleotide,” refers to a short length ofsingle-stranded polynucleotide chain. Oligonucleotides are typicallyless than 200 residues long (e.g., between 15 and 100), however, as usedherein, the term is also intended to encompass longer polynucleotidechains. Oligonucleotides are often referred to by their length. Forexample a 24 residue oligonucleotide is referred to as a “24-mer”.Oligonucleotides can form secondary and tertiary structures byself-hybridizing or by hybridizing to other polynucleotides. Suchstructures can include, but are not limited to, duplexes, hairpins,cruciforms, bends, and triplexes.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, the sequence“5′-A-G-T-3′,” is complementary to the sequence “3′-T-C-A-5′.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. This is of particular importance inamplification reactions, as well as detection methods that depend uponbinding between nucleic acids.

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). A partiallycomplementary sequence is a nucleic acid molecule that at leastpartially inhibits a completely complementary nucleic acid molecule fromhybridizing to a target nucleic acid is “substantially homologous.” Theinhibition of hybridization of the completely complementary sequence tothe target sequence may be examined using a hybridization assay(Southern or Northern blot, solution hybridization and the like) underconditions of low stringency. A substantially homologous sequence orprobe will compete for and inhibit the binding (i.e., the hybridization)of a completely homologous nucleic acid molecule to a target underconditions of low stringency. This is not to say that conditions of lowstringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second target that issubstantially non-complementary (e.g., less than about 30% identity); inthe absence of non-specific binding the probe will not hybridize to thesecond non-complementary target.

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described above.

A gene may produce multiple RNA species that are generated bydifferential splicing of the primary RNA transcript. cDNAs that aresplice variants of the same gene will contain regions of sequenceidentity or complete homology (representing the presence of the sameexon or portion of the same exon on both cDNAs) and regions of completenon-identity (for example, representing the presence of exon “A” on cDNA1 wherein cDNA 2 contains exon “B” instead). Because the two cDNAscontain regions of sequence identity they will both hybridize to a probederived from the entire gene or portions of the gene containingsequences found on both cDNAs; the two splice variants are thereforesubstantially homologous to such a probe and to each other.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe that can hybridize(i.e., it is the complement of) the single-stranded nucleic acidsequence under conditions of low stringency as described above. As usedherein, the term “hybridization” is used in reference to the pairing ofcomplementary nucleic acids. Hybridization and the strength ofhybridization (i.e., the strength of the association between the nucleicacids) is impacted by such factors as the degree of complementarybetween the nucleic acids, stringency of the conditions involved, theT_(m) of the formed hybrid, and the G:C ratio within the nucleic acids.A single molecule that contains pairing of complementary nucleic acidswithin its structure is said to be “self-hybridized.”

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecomponent or contaminant with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is such present in a form orsetting that is different from that in which it is found in nature. Incontrast, non-isolated nucleic acids as nucleic acids such as DNA andRNA found in the state they exist in nature. For example, a given DNAsequence (e.g., a gene) is found on the host cell chromosome inproximity to neighboring genes; RNA sequences, such as a specific mRNAsequence encoding a specific protein, are found in the cell as a mixturewith numerous other mRNAs that encode a multitude of proteins. However,isolated nucleic acid encoding a given protein includes, by way ofexample, such nucleic acid in cells ordinarily expressing the givenprotein where the nucleic acid is in a chromosomal location differentfrom that of natural cells, or is otherwise flanked by a differentnucleic acid sequence than that found in nature. The isolated nucleicacid, oligonucleotide, or polynucleotide may be present insingle-stranded or double-stranded form. When an isolated nucleic acid,oligonucleotide or polynucleotide is to be utilized to express aprotein, the oligonucleotide or polynucleotide will contain at a minimumthe sense or coding strand (i.e., the oligonucleotide or polynucleotidemay be single-stranded), but may contain both the sense and anti-sensestrands (i.e., the oligonucleotide or polynucleotide may bedouble-stranded).

As used herein, the term “purified” or “to purify” refers to the removalof components (e.g., contaminants) from a sample. For example,antibodies are purified by removal of contaminating non-immunoglobulinproteins; they are also purified by the removal of immunoglobulin thatdoes not bind to the target molecule. The removal of non-immunoglobulinproteins and/or the removal of immunoglobulins that do not bind to thetarget molecule results in an increase in the percent of target-reactiveimmunoglobulins in the sample. In another example, recombinantpolypeptides are expressed in bacterial host cells and the polypeptidesare purified by the removal of host cell proteins; the percent ofrecombinant polypeptides is thereby increased in the sample.

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include bloodproducts, such as plasma, serum and the like. Environmental samplesinclude environmental material such as surface matter, soil, water, andindustrial samples. Such examples are not however to be construed aslimiting the sample types applicable to the present invention.

As used herein, the term “subject” refers to organisms to be treated bythe methods of the present invention. Such organisms preferably include,but are not limited to, mammals (e.g., murines, simians, equines,bovines, porcines, canines, felines, and the like), and most preferablyincludes humans. In the context of the invention, the term “subject”generally refers to an individual who will receive or who has receivedtreatment (e.g., administration of a compound of the present inventionand optionally one or more other agents) for a condition characterizedby bacterial infection.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to compositions and methods for targetingexpression of exogenous genes to platelets. In particular, the presentdisclosure relates to treatment of hemophilia and other diseases andconditions by targeting expression of exogenous agents (e.g., clottingfactors) to platelets.

Embodiments of the present invention provide compositions and method fordirecting expression of a heterologous gene to a specific cell typeusing a cell-specific promoter. For example, in some embodiments,expression of heterologous or exogenous genes is targeted to a stem cell(e.g., cancer stem cell or hematopoietic stem cell) that in turnexpresses the gene of interest in progenitor cells (e.g., platelets).The compositions and methods find use in the treatment of a variety ofdisease (e.g., platelet mediated diseases such as hemophilia). Certainembodiments of the present invention are illustrated based on treatmentof hemophilia with exogenous or heterologous clotting factors, althoughthe present invention is not limited to the treatment of hemophilia orplatelet disorders.

In some embodiments, the present disclosure relates to treatment ofhemophilia and rare and common inherited bleeding disorders as well asother diseases states that involve platelets (e.g. thrombosis of veinsand arteries, immune response, and cancer) and conditions by employinghematopoietic stem cell gene therapy using a fragment of a plateletspecific gene promoter to drive expression of proteins only in theplatelet lineage and in some circumstances fusion of a signal peptide tothe therapeutic molecule to traffic recombinant proteins specifically toplatelet α-granules to induce regulated release of the exogenous agentsfrom activated platelets at the site of injury. In summary, thisapproach allows platelets to be utilized as a vehicle to delivertherapeutic agents to enable wound repair targeting expression ofexogenous agents (e.g., normal replacement proteins to restorehemostasis by correcting inherited platelet defects, clotting factorsfor hemophilia, anti-thrombotic agents for deep vein thrombosis andartery occlusion and anti-neoplastic agents to shrink solid tumors andprevent angiogenesis in cancer) to platelets.

There are several well-characterized inherited genetic defects thataffect various aspects of platelet function (activation, adhesion,aggregation, signal transduction, granule storage), which usuallymanifest themselves clinically as a failure to control bleeding (Leslie,M. Science 328, 562-564 (2010)). Embodiments of the present inventionprovide autologous transplant of hematopoietic stem cells transducedwith genes encoding normal integrin gene promoter driving synthesis ofcoagulation FVIII within platelets for correction of hemophilia A withinhumans. De novo synthesis of a biologically normal molecule withinmegakaryocytes has previous allowed trafficking of the entire protein toallow platelets to participate in wound repair. This is supported by therecent success of using hematopoietic stem cell gene transfer ofintegrin αIIb gene promoter driving expression of integrin αIIb togenerate denovo synthesis of integrin αIIbβ3 receptor complex onplatelets for improved platelet function and reduced bleeding times andblood loss for dogs deficient in integrin αIIb affected with canineGlanzmann Thrombasthenia (GT) (Fang, J. et al. Proc Natl Acad Sci USA108, 9583-9588 (2011)).

Transduction of G-CSF mobilized peripheral blood stem cells (G-PBC) withan oncoretrovirus vector encoding integrin (33 generated de novosynthesis of viable integrin αIIbβ3 complexes on megakaryocytes derivedfrom human GT patients (Wilcox, D. A et al., Blood. 95: 3645-52, 2000;Leslie, M. Science 328, 562-564 (2010)). It has also been shown thatplatelet function could be corrected within a murine model for GT bytransplantation of bone marrow transduced with a lentivirus vectorencoding (33 (Fang, J. et al., Blood 106, 2671-2679 (2005)) and that theuse of hematopoietic stem cell gene transfer of integrin αIIb togenerate αIIbβ3 on platelets can correct Canine Glanzmann Thrombasthenia(GT) (Fang, J. et al. Proc Natl Acad Sci USA 108, 9583-9588 (2011)).

Experiments conducted during the course of development of embodiments ofthe present invention demonstrate transferring genes into G-PBC showthat oncoretrovirus transduced human megakaryocytes and platelets couldsynthesize and store human coagulation factor VIII and release FVIIIupon activation in vitro with physiological agonists of plateletactivation (Wilcox, D. A., et al., Journal of Thrombosis andHaemostasis. 1: 2477-89, 2003). It has also been shown that hemostasiscould be improved within a murine model for hemophilia A (even in thepresence of inhibitory antibodies to FVIII) by transplantation of bonemarrow transduced with a lentivirus vector under the transcriptionalcontrol of the −889 fragment of the integrin αIIb gene promoter drivingexpression of human FVIII (Shi, Q. and Wilcox, D. A., et al., Journal ofThrombosis and Haemostasis 5: 352-361, 2007) and (Shi, Q., et al., Blood112: 2713-21, 2008) and that the use of G-CSF mobilized PBC forhematopoietic stem cell lentivirus mediate gene transfer of integrinαIIb gene promoter fragment with and without VWFSPD2 fused to humanFVIII induced expression of fully functional FVIII within platelets andplatelet α granules, which corrected platelet function and resulted inimproved hemostasis and the reduction and/or absence of any seriousbleeding events; thus animals did not require injection with FVIIIprotein replacement therapy within a large animal “canine” model forhemophilia A for at least 2.5 years after transplant as well asinhibitory antibodies were not generated to the recombinant human FVIIIstored within canine platelets

Accordingly, in some embodiments the present invention providescompositions and methods for genetic therapies for targeting stem cells(e.g., hematopoietic stem cells). The compositions and methods describedherein find use in the treatment of a variety of disease and conditions(e.g., platelet mediated disorders).

Some embodiments of the present invention are illustrated with thetreatment of hemophilia and other platelet diseases, although thepresent invention is not limited to the treatment of hemophilia. In someembodiments, compositions and methods comprise in vivo or ex vivogenetic therapies. For example, in some embodiments, hematopoietic stemcells are mobilized and targeted ex vivo with vectors that targetexpression of exogenous genes (e.g., clotting factors, platelet proteinspertinent to platelet function, anti-thrombotic agents for thromboticdisorders and anti-angiogenic and anti-neoplastic agents for oncogenicdisorders) specifically to platelets following reintroduction of themodified hematopoietic stem cells.

I. Vectors

The present invention is not limited to a particular targeting vector orexpression cassette. In some embodiments, expression cassettes comprisean exogenous gene of interest operable linked to a cell (e.g., platelet)specific promoter. In some embodiments, expression cassettes furthercomprise genes expression targeting or signal molecules, as well asexpression enhancers. FIGS. 7, 8, and 10 show exemplary expressioncassettes and vectors useful in embodiments of the present invention.Exemplary vector components are described below.

A. Promoters

In some embodiments, vectors comprise promoters that direct geneexpression to particular cell type. For example, in some embodiments,promoters are platelet specific promoters. The present invention is notlimited to particular platelet specific promoter. In some embodiments,truncated integrin αIIb gene (ITGA2B) promoters are used. Exemplarypromoters include, but are not limited to, −1218, −889 and −673 ITGA2Bpromoters that encode“ETS” and “GATA” elements for high level of genetranscription within megakaryocytes and a Repressor region that inhibitsgene transcription within other hematopoietic cell lineages (SEQ ID NOs:21, 22, 23; and 25; FIGS. 8 and 10).

The nucleotide sequence of the ITGA2B gene promoter was firstcharacterized in 1988 by a group in France headed by Dr. GerardMarguerie (Prandini M R, Denarier E, Frachet P, Uzan G, Marguerie G.Isolation of the human platelet glycoprotein IIb gene andcharacterization of the 5′ flanking region. Biochem Biophys Res Commun1988, 156(1): 595-601). FIG. 7 shows structural regions of the ITGA2Bpromoter. In some embodiments, a fragment (e.g., 1218, −889 and −673) ofthe promoter is utilized. In some embodiments, the −673 contains all ofthe essential regulatory elements to drive platelet-specific transgeneexpression. In 1993, researchers perform gene promoter studies and foundthat the transcription factor GATA-1 was important for high level geneexpression in megakaryocytes with at least three and maybe a fourthregion serves as GATA-1 binding site identified in ITGA2B (Martin F,Prandini M R, Thevenon D, Marguerie G, Uzan G. The transcription factorGATA-1 regulates the promoter activity of the platelet glycoprotein IIbgene. J Biol Chem 1993, 268(29): 21606-21612). Next it was discoveredthat there are three consensus sequences for another transcriptionfactor Ets to bind to ITGA2B that are believed to act together toproduce a high level of transgene expression (Lemarchandel V, GhysdaelJ, Mignotte V, Rahuel C, Romeo P H. GATA and Ets cis-acting sequencesmediate megakaryocyte-specific expression. Mol Cell Biol 1993, 13(1):668-676). The Murine ITGA2B promoter was cloned and found to have veryhigh nucleotide sequence homology with the human ITGA2B promoter,especially at the regions where transcription factors consensus bindingsequences were identified (Denarier E, Martin F, Martineau S, MarguerieG. PCR cloning and sequence of the murine GPIIb gene promoter. BiochemBiophys Res Commun 1993, 195(3): 1360-1364). Further gene promoteranalysis showed that a region of ITGA2B from −139 to −63 must bepreserved to prevent the gene promoter from driving transgenetranscription within other hematopoietic lineages, thus this region islabeled the Repressor (Prandini M H, Martin F, Thevenon D, Uzan G. Thetissue-specific transcriptional regulation of the megakaryocyticglycoprotein IIb gene is controlled by interactions between a repressorand positive cis-acting elements. Blood 1996, 88(6): 2062-2070).Finally, a binding site for the Sp1 was identified in ITGA2B that isbelieved important to work with Ets in conjunction with the otherelements when promoter forms a three dimensional structure that isessential for optimal platelet specific gene transcription (Block K L,Shou Y, Poncz M. An Ets/Sp1 interaction in the 5′-flanking region of themegakaryocyte-specific alpha IIb gene appears to stabilize Sp1 bindingand is essential for expression of this TATA-less gene. Blood 1996,88(6): 2071-2080).

The present invention is not limited to the ITGA2B promoters describedin SEQ ID NOs: 21-23). Embodiments of the present invention contemplatefragments, portions, and combinations of the fragments described herein.In some embodiments, fragments are shorter than the full length ITGA2Bpromoter (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 75,100, 150, 200 or more nucleotides shorter) and maintain desired activity(e.g., the ability to drive cell-specific expression to elicit a desiredeffect, e.g., reduction in sing or symptoms of a disease or condition).

In some embodiments, promoters comprise the ITGA2B fragments describedin SEQ ID NOs: 21, 22, and 23, or sequences that are 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 60, 75, 100, 150, 200, 300, 400, 500, 600or more nucleotides larger or smaller than SEQ ID NOs: 21, 22, and 23.For example, in some embodiments, fragments that are 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 60, 75, 100, 150, 200 or more nucleotidessmaller than SEQ ID NO:21 are utilized. In some embodiments, fragmentsthat are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 75, 100,150, 200, 300, 400, 500, 600 or more nucleotides larger than SEQ IDNO:23 are utilized.

In some embodiments, discontinuous fragments of SEQ ID NOs: 21, 22, or23 that retain promoter activity are utilized. For example in someembodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 75, 100,150, 200, 300, 400, 500, 600 nucleotides of SEQ ID NOs 21, 22, or 23 areutilized.

In some embodiments, promoter fragments comprise one or more elementsuseful for promoter activity. Examples include, but are not limited to,GATA elements (e.g., GATA54 or GATA454), sP1 elements, Ets35 elements,and the like (See e.g., Block et al., Blood 1994 84: 3385-3393; Prandiniet al., Blood 1996 88: 2062-2070; Block et al., Blood 1996 88:2071-2080; and Doubeikovski et al., J. Biol. Chem. 272: 24300-24307,1997; each of which is herein incorporated by reference in itsentirety).

In some embodiments, the 5′ ends of promoters are modified to addrestriction endonuclease sites to aid in cloning and constructingexpression vectors. For example, in some embodiments, 1, 2, 3, or 4nucleotides at the 5′ end are modified from the wild type sequence orthe fragments disclosed herein to add restriction endonuclease sites.

In some embodiments, the fragments comprise, consist essentially of, orconsist of promoter sequence found in SEQ ID NOs: 21, 22, or 23.

B. Heterologous Genes

The present invention is not limited to a particular exogenous gene. Inembodiments that treat hemophilia, exogenous genes are generallyclotting factors (e.g., Factor VIII and/or Factor IX). Human Factor VIIIhas the accession number NM_000132.3 and Human Factor IX has theaccession number NM_000133.3.

Other exogenous genes may be utilized in the treatment of other plateletrelated conditions. In some embodiments, exogenous genes useful in thetreatment of diseases other than platelet related disorders are utilized(e.g., in the treatment of cancer by using platelets to target releaseof anti-neplastic agents “i.e. IL-24” to shrink solid tumors andanti-thrombotic agents to be released at the site of blood clots such ascases of deep vein thrombosis).

C. Targeting Factors and Enhancers

In some embodiments, expression cassettes further comprise a targetingfactor that targets expression into a particular sub-structure of aplatelet. In some embodiments, expression cassettes further comprise theminimal amino acid sequence of a signal sequence peptide that has beenfound to be able traffic not only VWF but also recombinant proteinsfused to the peptide that has the proven ability to store proteins incellular granule compartments specifically the natural storagesub-structure endothelial cells “Weibel-Palade bodies” and plateletα-granules, both of which can be secreted upon cellular activation. Forexample, in some embodiments, expression constructs comprise a nucleicacid encoding a Von Willebrand Factor propeptide signal peptide and D2domain (SPD2) to promote trafficking of molecules directly into theα-granule compartment as shown in Rosenberg J B et al, IntracellularTrafficking of FVIII to von Willebrand Factor storage Granules, J. Clin.Invest. 101, 613-624 (1998); Haberichter S L, Jacobi P, Montgomery R R.Critical independent regions in the VWF propeptide and mature VWF thatenable normal VWF storage. Blood 2003, 101(4): 1384-1391 and Haberichteret al, The Von Willlebrand Factor Propeptide (VWFpp) Traffics anUnrelated Protein to Storage, Arterioscler Thromb Vas Biol. 22, 921 926(2002).

An example of an expression cassette comprising a 673 bp fragment of theITGA2B gene promoter and a VWF/SPD2 gene is shown in FIG. 7 (SEQ IDNO:24). Variants of these sequences that retain its desired activity arespecifically contemplated for use in compositions and methods ofembodiments of the present invention.

In some embodiments, constructs comprise an expression enhancer (e.g.,Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element(WPRE)) between the promoter/signaling cassette and the exogenous geneof interest. This element has been utilized by several gene transferstrategies because its structure inhibits degradation of the transcriptwithin the cell and thus allows for more therapeutic protein to besynthesized compared to a gene transfer vector in the absence of WPRE.

D. Vector Backbones

The present invention is not limited to a particular expression vector.In some embodiments, vectors are self-inactivating. In some embodiments,vectors are retroviral vectors (e.g., lentiviral vectors). Table 2provides a summary of exemplary suitable vectors.

TABLE 2 Virus Advantages Disadvantages Adenovirus High titer ImmunogenicHigh gene expression Does not integrate into genome Can infectnon-dividing cells Accepts very large cassettes (40 kb) Adeno-associatedvirus Can infect non-dividing cells Accepts small cassettes (4 kb)Relatively safe in humans Low transduction efficiency in hematopoieticcells Alphavirus (Sindbis) Can infect non-dividing cells Toxic to cellsHigh titer Does not integrate into genome High transduction efficiencyHigh gene expression Lentivirus Stably incorporated into genome New tofield Can infect non-dividing cells Safety uncertain in humansRetrovirus Stably incorporated into genome Infects only dividing cellsRelatively safe in humans High titer Accepts large cassettes (8 kb)

Retroviruses (family Retroviridae) are divided into three groups: thespumaviruses (e.g., human foamy virus); the lentiviruses (e.g., humanimmunodeficiency virus and sheep visna virus) and the oncoviruses (e.g.,MLV, Rous sarcoma virus).

Retroviruses are enveloped (e.g., surrounded by a host cell-derivedlipid bilayer membrane) single-stranded RNA viruses that infect animalcells. When a retrovirus infects a cell, its RNA genome is convertedinto a double-stranded linear DNA form (e.g., it is reversetranscribed). The DNA form of the virus is then integrated into the hostcell genome as a provirus. The provirus serves as a template for theproduction of additional viral genomes and viral mRNAs. Mature viralparticles containing two copies of genomic RNA bud from the surface ofthe infected cell. The viral particle comprises the genomic RNA, reversetranscriptase and other pol gene products inside the viral capsid (whichcontains the viral gag gene products), which is surrounded by a lipidbilayer membrane derived from the host cell containing the viralenvelope glycoproteins (also referred to as membrane-associatedproteins).

The organization of the genomes of numerous retroviruses has allowed theadaptation of the retroviral genome to produce retroviral vectors. Theproduction of a recombinant retroviral vector carrying a gene ofinterest is typically achieved in two stages. First, the gene ofinterest is inserted into a retroviral vector which contains thesequences necessary for the efficient expression of the gene of interest(including promoter and/or enhancer elements which may be provided bythe viral long terminal repeats [LTRs] or by an internalpromoter/enhancer and relevant splicing signals), sequences required forthe efficient packaging of the viral RNA into infectious virions (e.g.,the packaging signal [Psi], the tRNA primer binding site [-PBS], the 3′regulatory sequences required for reverse transcription [+PBS] and theviral LTRs). The LTRs contain sequences required for the association ofviral genomic RNA, reverse transcriptase and integrase functions, andsequences involved in directing the expression of the genomic RNA to bepackaged in viral particles. For safety reasons, many recombinantretroviral vectors lack functional copies of the genes that areessential for viral replication (these essential genes are eitherdeleted or disabled); the resulting virus is said to be replicationdefective.

Second, following the construction of the recombinant vector, the vectorDNA is introduced into a packaging cell line. Packaging cell linesprovide viral proteins required in trans for the packaging of the viralgenomic RNA into viral particles having the desired host range (e.g.,the viral-encoded gag, pol and env proteins). The host range iscontrolled, in part, by the type of envelope gene product expressed onthe surface of the viral particle. Packaging cell lines may expressecotrophic, amphotropic or xenotropic envelope gene products.Alternatively, the packaging cell line may lack sequences encoding aviral envelope (env) protein. In this case the packaging cell line willpackage the viral genome into particles that lack a membrane-associatedprotein (e.g., an env protein). In order to produce viral particlescontaining a membrane associated protein that will permit entry of thevirus into a cell, the packaging cell line containing the retroviralsequences is transfected with sequences encoding a membrane-associatedprotein (e.g., the G protein of vesicular stomatitis virus [VSV]). Thetransfected packaging cell will then produce viral particles thatcontain the membrane-associated protein expressed by the transfectedpackaging cell line; these viral particles, which contain viral genomicRNA derived from one virus encapsidated by the envelope proteins ofanother virus are said to be pseudotyped virus particles.

Viral vectors, including recombinant retroviral vectors, provide a moreefficient means of transferring genes into cells as compared to othertechniques such as calcium phosphate-DNA co-precipitation orDEAE-dextran-mediated transfection, electroporation or microinjection ofnucleic acids. It is believed that the efficiency of viral transfer isdue in part to the fact that the transfer of nucleic acid is areceptor-mediated process (i.e., the virus binds to a specific receptorprotein on the surface of the cell to be infected). In addition, thevirally transferred nucleic acid once inside a cell integrates incontrolled manner in contrast to the integration of nucleic acids whichare not virally transferred; nucleic acids transferred by other meanssuch as calcium phosphate-DNA co-precipitation are subject torearrangement and degradation.

Commonly used recombinant retroviral vectors are derived from theamphotropic Moloney murine leukemia virus (MoMLV) (Miller and Baltimore,Mol. Cell. Biol., 6:2895 [1986]). The MoMLV system has severaladvantages: 1) this specific retrovirus can infect many different celltypes, 2) established packaging cell lines are available for theproduction of recombinant MoMLV viral particles and 3) the transferredgenes are permanently integrated into the target cell chromosome. Theestablished MoMLV vector systems comprise a DNA vector containing asmall portion of the retroviral sequence (the viral long terminal repeator “LTR” and the packaging or “psi” signal) and a packaging cell line.The gene to be transferred is inserted into the DNA vector. The viralsequences present on the DNA vector provide the signals necessary forthe insertion or packaging of the vector RNA into the viral particle andfor the expression of the inserted gene. The packaging cell lineprovides the viral proteins required for particle assembly (Markowitz etal., J. Virol., 62:1120 [1988]).

Despite these advantages, existing retroviral vectors based upon MoMLVare limited by several intrinsic problems: 1) they do not infectnon-dividing cells (Miller et al., Mol. Cell. Biol., 10:4239 [1992]), 2)they produce low titers of the recombinant virus (Miller and Rosman,BioTechn., 7: 980 [1989]; and Miller, Nature 357: 455 [1992]) and 3)they infect certain cell types (e.g., human lymphocytes) with lowefficiency (Adams et al., Proc. Natl. Acad. Sci. USA 89:8981 [1992]).The low titers associated with MoMLV-based vectors has been attributed,at least in part, to the instability of the virus-encoded envelopeprotein. Concentration of retrovirus stocks by physical means (e.g.,ultracentrifugation and ultrafiltration) leads to a severe loss ofinfectious virus.

Other commonly used retrovectors are derived from lentivirusesincluding, but not limited to, human immunodeficiency virus (HIV) orfeline immunodeficiency virus (Hy). Lentivirus vectors have theadvantage of being able to infect non replicating cells.

The low titer and inefficient infection of certain cell types by retrovectors has been overcome by the use of pseudotyped retroviral vectorswhich contain the G protein of VSV as the membrane associated protein.Unlike retroviral envelope proteins which bind to a specific cellsurface protein receptor to gain entry into a cell, the VSV G proteininteracts with a phospholipid component of the plasma membrane(Mastromarino et al., J. Gen. Virol., 68:2359 [1977]). Because entry ofVSV into a cell is not dependent upon the presence of specific proteinreceptors, VSV has an extremely broad host range. Pseudotyped retroviralvectors bearing the VSV G protein have an altered host rangecharacteristic of VSV (i.e., they can infect almost all species ofvertebrate, invertebrate and insect cells). Importantly, VSVG-pseudotyped retroviral vectors can be concentrated 2000-fold or moreby ultracentrifugation without significant loss of infectivity (Burns etal., Proc. Natl. Acad. Sci. USA, 90:8033 [1993]).

The VSV G protein has also been used to pseudotype retroviral vectorsbased upon the human immunodeficiency virus (HIV) (Naldini et al.,Science 272:263 [1996]). Thus, the VSV G protein may be used to generatea variety of pseudotyped retroviral vectors and is not limited tovectors based on MoMLV.

The present invention is not limited to the use of the VSV G proteinwhen a viral G protein is employed as the heterologousmembrane-associated protein within a viral particle. Sequences encodingother G proteins derived from other members of the Rhabdoviridae familymay be used; sequences encoding numerous rhabdoviral G proteins areavailable from the GenBank database.

The majority of retroviruses can transfer or integrate a double-strandedlinear form of the virus (the provirus) into the genome of the recipientcell only if the recipient cell is cycling (i.e., dividing) at the timeof infection. Retroviruses that have been shown to infect dividing cellsexclusively, or more efficiently, include MLV, spleen necrosis virus,Rous sarcoma virus human immunodeficiency virus, and other lentiviralvectors.

It has been shown that the integration of MLV virus DNA depends upon thehost cell's progression through mitosis and it has been postulated thatthe dependence upon mitosis reflects a requirement for the breakdown ofthe nuclear envelope in order for the viral integration complex to gainentry into the nucleus (Roe et al., EMBO J., 12:2099 [1993]). However,as integration does not occur in cells arrested in metaphase, thebreakdown of the nuclear envelope alone may not be sufficient to permitviral integration; there may be additional requirements such as thestate of condensation of the genomic DNA (Roe et al., supra).

The present invention is not limited to retroviral vectors. Largenumbers of suitable vectors are known to those of skill in the art, andare commercially available. Such vectors include, but are not limitedto, the following vectors: 1) Bacterial—pQE70, pQE60, pQE 9 (Qiagen),pBS, pD10, phagescript, psiX174, pbluescript SK, pBSKS, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223 3, pKK233 3, pDR540, pRIT5(Pharmacia); and 2) Eukaryotic—pWLNEO, pSV2CAT, pOG44, PXT1, pSG(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). Any other plasmid orvector may be used as long as they are replicable and viable in thehost. In some embodiments of the present invention, mammalian expressionvectors comprise, along with an expression cassette as described herein,an origin of replication, any necessary ribosome binding sites,polyadenylation sites, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking non transcribed sequences. Inother embodiments, DNA sequences derived from the SV40 splice, andpolyadenylation sites may be used to provide the non-transcribed geneticelements.

II. Therapeutic Methods

In some embodiments, the present invention provides systems and methodsfor genetic manipulation of stem cells (e.g., hematopoietic stem cellsor cancer stem cells). In some embodiments, the compositions and methodsdescribed herein find use in the treatment of a variety of disorderrelated to platelet function (e.g., hemophilia and the disordersdescribed in Table 3 below).

There have been several bleeding disorders characterized by moleculargenetic defects of platelet membrane, cytoplasmic and granular proteinsthat usually lead to prolonged bleeding events. While each disorder israre, maybe occurring in 1:1,000,000 individuals (e.g. GlanzmannThrombasthenia), taken collectively, an inherited platelet defect occurin 1:20,000 people worldwide as described in Wilcox, D. A. White II, G.C, Gene therapy for platelet disorders: studies with glanzmann'sthrombasthenia. Journal of Thrombosis and Haemostasis. 1: 2300-2311,(2003) and Wilcox, D. A., White II, G. C: Gene therapy for plateletdisorders. In: Platelets. Second Edition, A. D. Michelson (ed.),Academic Press, San Diego, Chapter 71: 1313-1325, (2007) and ThirdEdition, Chapter 64: In Press (2012). In addition to inherited plateletdefects, hematopoietic stem cell gene therapy aimed at targetingtherapeutic agents to the platelet surface, cytoplasm or granules findsuse as a strategy to correct other disorders of hemostasis, thrombosis,immune response and cancer.

TABLE 3 Inherited Disorder Defect Function Disrupted G-Protein DisorderG_(αq), G_(αi1) Activation ADP Receptor Defect P2Y₁₂ ActivationBernard-Soulier Syndrome Glycoprotein Ib-IX Adhesion Collagen ReceptorDeficiency Glycoproteins Ia-IIa Adhesion Glanzmann ThrombastheniaGlycoproteins IIb-IIIa Aggregation Gray Platelet Syndrome NBEAL2α-Granule Formation/Storage QuebecPlatelet Disorder Urokinaseplasminogen α-Granule Storage activator Scott SyndromePhosphatidylserine Coagulation Translocation May-Hegglin Anomaly MYH9Gytoskeleton/Platelet Formation Fechtner Syndrome Sebastian PlateletSyndrome Epstein Syndrome Wiskott-Aldrich Syndrome WAS ProteinCytoskeleton Chediak-Higashi Syndrome CHS protein Dense BodyFormation/Storage Hermansky-Pudiak Syndrome HPS1, HPS3-7, AP-3 DenseBody Formation/Storage Thromboxane Deficiency Thromboxane A₂ SignalTransduction

In some embodiments, therapeutic methods are ex vivo methods, in whichautologous hematopoietic stem cells are harvested from an animal (e.g.,human) in need of treatment, modified using one of the vector describedherein, and re-introduced into the original donor. Such autologousmethods reduce the risk of autoimmune or rejection responses that canoccur with infusion of donor clotting factors and allow one to limitgene transduction to hematopoietic stem cells through ex vivotransduction.

An exemplary method for ex vivo modification of hematopoietic stem cellsis described in Aiuti et al. (Science 341:865 (2013; herein incorporatedby reference in its entirety). For example, in some embodiments, themethod includes the steps of administering cytokines to mobilizeperipheral blood stem cells into the peripheral blood; performingapheresis and magnetic bead selection for CD34+ cells; preconditioningusing e.g., bulsulfan and/or other agents like fludarabine; and using aviral (e.g., lentiviral) gene transfer vector to modify stem cellsbefore they are re-introduced into the patient.

In some embodiments, hematopoietic stem cells are mobilized usingadministration of cytokines or other mobilization agents (See e.g., Fuet al., Blood Rev. 2000 December; 14(4):205-18 and U.S. Pat. No.7,417,026, each of which is herein incorporated by reference in itsentirety for a discussion of mobilization protocols), although othersuitable protocols may be utilized.

For example, in some embodiments, mobilization cytokines include, butare not limited to, Interleukin-3 (IL-3), granulocyte colony stimulatingfactor (G-CSF), also known as Amgen's FDA approved drug Neupogen, stemcell factor (SCF), granulocyte macrophage colony-stimulating factor(GM-CSF), and sequential or co-administration of one or more of IL-3,GM-CSF, SCF, and GM-CSF.

Suitable dosage ranges for mobilization agents vary, but in general, thecompounds are administered in the range of about 0.1 μg/kg-5 mg/kg ofbody weight; preferably the range is about 1 μg/kg-300 μg/kg of bodyweight; more preferably about 10 μg/kg-100 μg/kg of body weight. For atypical 70-kg human subject, thus, the dosage range is from about 0.7μg-350 mg; preferably about 700 μg-21 mg; most preferably about 700 μg-7mg. Dosages can be higher when the compounds are administered orally ortransdermally as compared to, for example, i.v. administration. Thecompounds can be administered as a single bolus dose, a dose over time,as in i.v. or transdermal administration, or in multiple dosages.

The amount of active compound to be administered can vary according tothe discretion of the skilled artisan. The amount of active compound tobe administered to the recipient is within the ranges described abovefor stem cell mobilization. However, the administration of such amountswill vary according to the standards set forth by clinicians in thefield of stem cell enhancement therapy.

Following mobilization, CD34+ Peripheral Blood stem cells (PBC) areisolated from the low molecular weight mononuclear cells byimmunomagnetic beads using Miltenyi's automacs system (for large animal,dogs, 25-45 kg) and Miltenyi's Clinimacs system (for humans) recentlyapproved for clinical use by the FDA. The CD34+PBC are the geneticallymodified using the vectors described herein and re-introduced into asubject in need by autologous stem cell transplant. In some embodiments,a single treatment is utilized to provide long-term protection againstepisodes of bleeding. In some embodiments that treat hemophilia,treatment is performed on a regular basis (e.g., weekly, monthly,yearly, once every 2, 3, 4, 5 or more years, and the like) in order toprevent episodes of bleeding. In some embodiments, treatment is onlyadministered when episodes of abnormal bleeding occur (e.g., followingaccidents, prior to or following surgery, etc,). In some embodiments,maintenance therapy is administered in combination with extra therapywhen episodes of abnormal bleeding occur.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1 Materials and Methods Cell Lines.

Human transformed cell lines were obtained from American Type CultureCollection (Rockville, Md.) and propogated under conditions describedfor promegakaryocytic (HEL Megakaryocyte transformed cell line) (Bray,P. F. et al. J Clin Invest 80, 1812-1817. (1987); Greenberg, S. M., etal., T-cell lymphoma (KT1), (Okamoto, T., et al. J Biol Chem 261,4615-4619 (1986)) B-cell lymphoma (Raji), (Choi, J. H. et al.International immunopharmacology 8, 852-858, 2008.01.037 (2008))Erythroleukemia (K562), (Gauwerky, C. & Golde, D. W. Blood 56, 886-891(1980)) and Epithelial (HeLa) (Goldstein, M. N., et al., Annals of theNew York Academy of Sciences 89, 474-483 (1960)) cell lines.

Luciferase Reporter Gene Promoter Vectors.

ITGA2B Gene Promoter Constructs: Genomic DNA was isolated from the humanpro-megakaryocyte cell line, Dami, 49 and human ITGA2B gene promoterfragments were amplified by PCR using either sense primer “−1218”(5′-TTACGCGTCGACAGATCTAAATGTGGCTGGTTACCCC-3′)“−1198” (SEQ ID NO:1)(bold) of ITGA2B or “−889”(5′-TTACGCGTCGACAGATCTGTGCTCAATGCTGTGCC-3′)“−872” (SEQ ID NO:2) (bold)of ITGA2B, or “−673” (5′-TTACGCGTCGACAGATCTCCTTGCCACCTAGACC-3′)“−654”(SEQ ID NO:3) (bold) of ITGA2B and anti-sense primer(5′-GGCGTCTTCCATGGTCCTTCTTCCACAACC-3′) (SEQ ID NO:4) encodingnucleotides +99 to +86 of luciferase pGL3-BASIC and nucleotides +30 to+15 (bold) of ITGA2B gene promoter. Correct identity of constructs wasconfirmed by nucleotide sequence analysis.

pCMVLuc: A BglII and HindIII restriction digest of cytomegalovirustissue non-specific gene promoter (878 bp) from pRc/CMV (Invitrogen) isligated into the pGL3-Basic Luciferase vector (Promega, Madison, Wis.).This construct served as the positive control for high level geneexpression within all cell-types; thus, assigned an arbitrary level of100% luciferase activity for each cell line (FIG. 1).

pGL3-BasicLuc: Negative control construct for 0% luciferase activity(FIG. 1) because lacks a gene promoter to drive luciferase genetranscription (Promega).

pCMVnlac: Cell lines were co-transfected with one of the pITGA2BLuc+constructs and pCMVnlac encoding the β-galactosidase marker gene tonormalize transgene expression (Wilcox et al., Proc Natl Acad Sci USA1999, 96(17): 9654-9659.

Luciferase Gene Promoter Reporter Assay.

Cell lines (2×10⁷) were co-transfected with either (20)μg) of the ITGA2Bgene promoter construct (−1218, −889, −673) (FIG. 1A) or the positive(CMV) or negative (Basic) controls encoding firefly luciferase andpCMVnlac (20 μg) encoding β-galactosidase.49 Briefly, forty-eight hoursafter co-transfection cells were washed, harvested, and lysates wereprepared and frozen to −80° C. using the luciferase assay system(Promega). Luciferase activity was measured with a Turner Designs Model20 Luminometer. Detection of β-galactosidase activity was performed tonormalize transient transgene expression for each cell line with asensitive ELISA enzymatic assay that measured colormetric change withthe substrate for β-galactosidase, chlorophenol redβ-D-galactophranoside (CPRG) (Eustice D C, et al., Biotechniques 1991,11(6): 739-740, 742-733). The percent of luciferase activity wasdetermined by comparing the mean value of the Relative Light Units (RLU)of luciferase/CPRG Vmax value for each construct to reveal thetransfection efficiency for each cell line. The RLU for pCMVLuc wasassigned arbitrarily a value of 100% and all other results werecalculated for each vector based upon that value as shown in FIG. 1A.

ITGA2B Promoter Driven Lentiviral Vector for Human BDDFVIII.

ITGA2B-(M)WPTS genetic transfer vectors are derived from a HIV type-1lentiviral vector (D.Trono, University of Geneva, Switzerland).51p-889ITGA2B-BDDFVIII-WPTS lentiviral vector (FIG. 1B) encodes a −889 to+30 nucleotide fragment of the human ITGA2B promoter and human BDDFVIIImolecule.16 p-673ITGA2B-VWFSPD2-BDDFVIII-WPTS lentiviral vector (FIG.1C) encodes a fragment of the human ITGA2B gene promoter from nucleotide−673 to +30 followed by a fragment of the human Von Willebrand Factorpropeptide (VWFpp encoding 540 amino acid VWF signal peptide (SP;66 bp)linked to the D2 domain (1,199 bp) and cDNA encoding human BDDFVIII toallow megakaryocyte-specific transcription of a hybrid molecule thatuses the SPD2 peptide to traffic human BDDFVIII to plateletα-granules.22 cDNA encoding SP was amplified by PCR with forward Primer(P)1(5′GTTAATCGATATCTCCTTGCCACCTAGA3′) (SEQ ID NO:5), and reverseP2(5′AATCTGGCAGGAATCATGGTCCTTCTTCCACAACCT3′) (SEQ ID NO:6) and ligatedto D2 amplified by PCR using forwardP3(5′AGGTTGTGGAAGAAGGACCATGATTCCTGCCAGATTTGC3′) (SEQ ID NO:7) andreverse P4(5′CGTCTCGGCCCTTTTGCTGCCATGAGACAG3′) (SEQ ID NO:8). A nestedPCR linked ITGA2B promoter and VWFSPD2 with P5(5′ATCGATATCTCCTTGCCACCTA3′) (SEQ ID NO:9) and P4. p-8891TGA2-BDDFVIII-WPTS served as a templatefor PCR of cDNA encoding a fragment of BDDFVIII using forwardP7(5′CGTCTCAGGGCCACCAGAAGATACTACCT3′) (SEQ ID NO:10) and reverseP8(5′ACGCGTCTTCTCTACATACTAGTA3′) (SEQ ID NO:11) to synthesize cDNA thatligated directly to VWFD2. All PCR products were cloned into pCR-BluntII-TOPO (Life Technologies, Grand Island, N.Y.) using unique restrictionsites −6731TGA2B-SPD2(ClaI and BsmBI) ligated to 5′hBDDFVIII (BsmB1 andMluI) with 3′BDDFVIII (MluI and Spel). All fragments were cloned intopWPTS lentiviral vector and the correct identity confirmed by nucleotidesequence analysis. Recombinant virions were generated from three-plasmidtransient co-transfection followed by supernatant collection, 500-foldconcentration by centrifugation, and storage at −80° C. until utilized(Fang J, et al., Proc Natl Acad Sci USA 2011, 108(23): 9583-9588).Virion titer was determined by RT-PCR (Lizee G, et al., Hum Gene Ther2003, 14(6): 497-507). Replication-competent virions were confirmedabsent from stocks with marker rescue assays (Wilcox et al., Blood 2000,95(12): 3645-3652).

Dogs.

Cytokine mobilized CD34+G-PBC gene transfer and autologous transplantstudies using FVIII-Deficient dogs affected with hemophilia A(University of North Carolina, Chapel Hill, N.C.) (Lozier J N, et al.,Proc Natl Acad Sci USA 2002, 99(20): 12991-12996) were conducted andapproved by Institutional Animal Care and Use Committees of theUniversity of North Carolina and The Medical College of Wisconsin whichare both accredited facilities of the American Association forAccreditation of Laboratory Animal Care.

Canine CD34+G-PBC Isolation, Transduction, Transplantation.

19 Adult (1.25, 4.25, and 6.5 year old) FVIII-Deficient male dogs wereinjected daily with canine recombinant granulocyte colony stimulatingfactor (crG-CSF;10 μg/kg/d) and stem cell factor (crSCF; 5 μg/kg/d)(Amgen, Thousand Oaks, Calif.). G-PBC collection was performed on thethird day using a COBE Spectra Blood Cell Separator. Mononuclear G-PBCwere isolated with Fico-Paque Plus (GE Healthcare, Uppsala, Sweden).CD34+ G-PBC were selected with a biotin-conjugated-1H6 Ab (1 mg/ml)(Richard Nash, Fred Hutchinson Research Institute, Seattle, Wash.) andanti-biotin immuno-magnetic beads (1:5 dilution) on an Automacs magneticcell separator (Miltenyi Biotec Inc., Auburn, Calif.). CD34+G-PBC weretransduced with −889ITGA2B-BDDFVIII-WPTS or−673ITGA2B-VWFSPD2-BDDFVIII-WPT S lentiviral vector. Briefly, 4×106cells/well were seeded in a 6-well plate (Falcon-Becton Dickinson,Franklin Lakes, N.J.) coated with 20 μg/cm2 RetroNectin (Takara Shuzo,Otsu, Shiga, Japan) and incubated with 1.0×10⁴ ITGA2B-FVIIIlentivirions/cell in X-Vivo 10 containing 10% FCS, rhIL-3, rcaIL-6,rcaSCF, rhTPO and rhflk2/flt3 ligand. Approximately 3×106FVIII-transduced CD34+G-PBC/kg and 2×108 CD34(−)G-PBC were infused intothe cephalic vein of each autologous transplant recipientpre-conditioned with a non-myeloablative dose of 5-10 mg/kg Busulfex®.Transient immune suppression administered for ≈90 days after transplantwith 10 mg/kg/d cyclosporine (Gengraf®, Abbott Laboratories, NorthChicago, Ill.) and 8 mg/kg/d MMF (Table 1) (Fang et al., Proc Natl AcadSci USA 2011, 108(23): 9583-9588).

Blood Collection.

Blood was collected at preselected times into a vacutube containing 7.5%EDTA anticoagulant (Fang et al., supra). Blood cells were counted on aVet ABC hematology analyzer (scil animal care company, Gurnee, Ill.).Platelets were isolated with Fico/Lite™ (Atlanta Biologicals, Norcross,Ga.), washed with PBS and used directly for immunofluorescent flowcytometry or FVIII:C activity analysis. Leukocytes were isolated withFicoll-Paque Plus® (GE Healthcare) according to the manufacturesspecifications.

Antibodies.

A murine monoclonal 1° Ab to canine CD34 “1H6” (1 mg/ml) was from theFred Hutchinson Cancer Research Center (Seattle, Wash.).27 A sheepanti-rabbit fibrinogen polyclonal 1° Ab (5 μg/ml) that recognizes caninefibrinogen was purchased from (Enzyme Research). Monoclonal 1° Abs (5-10μg/ml), MBC 103.3 and 301.3 (R. R. Montgomery, BloodCenter of WI,Milwaukee, Wis.), recognize epitopes on human BDDFVIII.53 2° Abs usedwere Alexa Fluor® 488 F(ab′)2 conjugated to a fragment of donkeyanti-sheep IgG (H+L) (1:1,000 dilution) and Alexa Fluor® 568 F(ab′)2fragment of goat anti-mouse IgG (H+L) (1:500 dilution) were from LifeTechnologies (Grand Island, N.Y.).

Immunofluorescent Confocal Microscopy.

Canine platelets were fixed with 3.7% (vol/vol) buffered formalin,permeabilized in 0.5% Triton X-100 (in 20 mmol/L Hepes, 300 mmol/Lsucrose, 50 mmol/L NaCl, and 3 mmol/L MgCl2, pH7.0), and blocked with2.5% normal goat serum in HBSS. Platelets were incubated with a sheeppolyclonal 1° Ab to canine fibrinogen and monoclonal 1° Ab (MBC 103.3 &301.3) to human FVIII (5 ug/ml) overnight at 4° C.53 The Alexa Fluor®488-conjugated F(ab′)2 fragment of donkey anti-sheep IgG (H+L) was usedas a 2° Ab (1:1,000 dilution) to detect fibrinogen and Alexa Fluor®568-conjugated F(ab′)2 fragment of goat anti-mouse IgG (H+L) conjugated2° Ab (1:500 dilution) was used to detect the presence of FVIII for 30min at 25° C. Platelets were mounted with Vectashield (Vector Labs,Burlingame, Calif.). Immunofluorescence was detected with a Zeiss LSM510 Multiphoton Confocal Microscope (Carl Zeiss, Inc. Oberkochen,Germany) (Wilcox D A, et al., Journal of thrombosis and haemostasis: JTH2003, 1(11): 2300-2311). Platelets isolated from FVIII-Deficient dogswere used as negative controls. Nonspecific isotype control Ab served asnegative controls. Platelets were imaged by Z sections taken for eachfield and the entire Z series (12-25 images) combined into a stackedprojection. The projections were merged using the Confocal Assistantsoftware program (Bio-Rad). Computer-assigned colors were based on theintensities of bitmap overlaps, with Alexa488-fluorochrome representedby green pixels, Alexa568-fluorochrome represented by red pixels, andco-localization of the two fluorochrome-conjugated antibodiesrepresented by yellow pixels.

Immunofluorescent Flow Cytometry.

Canine platelets were isolated from blood and treated with Cytofix™ andPERM/WASH™ reagents (BD Biosciences) for intracellular detection ofBDDFVIII. Platelets were incubated with a monoclonal 1° Ab (MBC 103.3 &301.3) to human FVIII (5 ug/ml) 30 minutes at 4° C. and then incubatedwith Alexa Fluor® 568-conjugated F(ab′)2 fragment of goat anti-mouse IgG(H+L) conjugated 2° Ab (1:500 dilution) for 30 minutes at 4° C.Platelets isolated from FVIII-Deficient dogs were used as negativecontrols. Nonspecific isotype control Ab served as negative controls.Cells were collected and analyzed on an Accuri® C6 Flow Cytometer(Accuri Cytometers, Inc., Ann Arbor Mich.) using the Accuri analysissoftware.

Immunogold Labeling.

Platelets were fixed in 1.25% glutaraldehyde (Fluka A G, Buchs,Switzerland), infused with 2.3M sucrose (Fluka), and frozen with aReichert K F 80 freezing system (Leica, Vienna, Austria). Sections of≈80 nm were prepared with the Ultracut E ultramicrotome equipped with aFC 4E cryokit attachment and placed on collodion-coated nickel grids.Grids were incubated for 10 min on PBS with 1% BSA and then placed on(10 μg/ml) drops of the 1° Ab to FVIII(301.3) for 1 h at 25° C. Sectionswere incubated for 1 h with a goat anti-mouse 2° Ab adsorbed onto 10 nmgold particles (1/100 dilution of AuroProbe EM G10). Controls includedthe use of an irrelevant IgG of the same species and at the sameconcentration.

Electron Microscopy.

Grids were stained by uranyl acetate and osmium and then embedded inmethylcellulose prior to observation with a Jeol JEM-1010 transmissionelectron microscope (Jeol, Croissy-sur-seine, France) at 80 KV.

Agonist Induced Activation of Platelets.

Platelets were isolated from circulating peripheral blood, washed, andactivated with physiological agonists of platelet activation. To induceactivation, platelets were resuspended in Tyrode's buffer (2.5×106/ml)containing 1 mM CaCl2, 1 mM MgCl2, 25 μM each of adenosine diphosphate(ADP) (Sigma), epinephrine (Bio/Data Corporation, Horsham, Pa.) andcanine thrombin receptor activating peptides: PAR1 (SFFLKN-NH2), PAR3(TRFGAP-NH2) and PAR4 (SFPGQP-NH2) for 30 minutes at 37° C. aspreviously described (Fang et al., supra). Separate aliquots wereincubated in Tyrode's buffer without agonist as a negative control. Theplatelets were pelleted by centrifugation and supernatant was aspiratedand discarded from agonist treated and negative control samples. Theplatelet pellet was frozen immediately to −80° C. until tested forFVIII:C activity using the coatest assay.

PCR Detection of Lentiviral Vector in Blood Genomic DNA.

DNA was isolated with a QIAamp® DNA Blood Mini Kit (Qiagen, Maryland,USA) from canine leukocytes purified with Ficoll-Paque™ Plus (AmershamPharmacia Biotech AB, Uppsala, Sweden). p-889ITGA2-BDDFVIII-WPTS servedas a positive control. PCR analysis was performed with Taq polymerase(Invitrogen, Carlsbad, Calif.) on a PTC200 instrument (MJ Research,Watertown, Mass.) with forward primer P1(5′-ACGCTATGTGGATACGCTG-3′) andreverse primer P2(5′-AACACCACGGAATTGTCAG-3′) (SEQ ID NO:12) tosynthesize a 318 nucleotide primary product encoding the WPRE (FIG.1B,C). A secondary PCR reaction was performed with nested forward primerP3(5′-TGGATACGCTGCTTTAATGC-3′) (SEQ ID NO:13) and reverse primerP4(5′-AATTGTCAGTGCCCAACAG-3′) (SEQ ID NO:14) encoding a 302 bp productof WPRE (FIG. 5A).

RT-qPCR to Detect Lentiviral Transduction Efficiency.

Percent lentiviral gene marking was measured by RT-qPCR using BIO-RADCFX96 Real-Time System.52 Briefly, 12.5 ul of TaqMan Universal PCRMaster Mix (Life technologies), a 900 nM concentration of each primer,and 200 nM probe were combined in 20 μl of water. Then 5 ul of caninegenomic DNA was added and PCR utilized 2 min at 500 C, 10 min at 950 C,and then 40 cycles of 15 sec at 950 C and 1 min at 600 C. For eachRT-qPCR, a no template control was included as negative control. Eachsample was analyzed in triplicate for gene copy number using PrimerExpress software (version 1.0; Applied Biosystems) and the mean valuefor transgene copy number/genome was converted to percent peripheralblood cells positive for lentiviral vector (also known as transductionefficiency) and reported in Table 1 Column 8. The lentiviral LTR primersand probe used were: Fwd:5′-AGCTTGCCTTGAGTGCTTCA-3′ (SEQ ID NO:15);Rev:5′-TGACTAAAAGGGTCTGAGGGA-3′(SEQ ID NO:16);probe:6FAM-TGCCCGTCTGTTGTGTGACTCTG-MGBNFQ (SEQ ID NO:17). The canineITGB3 gene was used as an endogenous control for gene copy number withFwd:5′-ATGCATCCCACTTGCTGGTAT-3′(SEQ ID NO:18);Rev:5′-TGCCCATCGTTAGGTTGG-3′(SEQ ID NO:19);probe:6FAM-TGCCTGCCAGCCTTCCATCCAG-MGBNFG (SEQ ID NO:20). Copy number wasbased on TaqMan principle. Ten-fold serial dilution of the plasmidconstructs of known concentration containing relevant sequences(Lentiviral vector LTR and canine ITGB3) were used to create standardcurves for quantification of samples.

Linear Amplification-Mediated (LAM)-PCR.

LAM-PCR was performed to localize the lentiviral vector insertion siteswithin genomic DNA isolated from peripheral blood leukocytes. Briefly,the junction between integrated proviral LTR and the host genome wasselected by 2 rounds of linear PCR [95° C. for 5 min; (95° C. for 1m,60° C. for 45s, 72° C. for 90s)×50; 72° C. for 10m] with avector-specific 5′-biotinylated primer[5′-/biotin/-GAACCCACTGCTTAAGCCTCA-3′(SEQ ID NO:26)] and purified usingstreptavidin-coated magnetic beads [Dynal M-280]. Products were doublestranded using Klenow polymerase and random hexanucleotide primers anddigested with Tsp509I at 65° C. for 2h. Directional double-strandedlinker oligos were ligated onto the non-LTR end and the resultingproducts were amplified by nested PCR [95° C. for 5 min; (95° C. for 1m, 60° C. for 45s, 72° C. for 90s)×35; 72° C. for 10m] usingLTR-specific forward primers [F1:5′-/biotin/-AGCTTGCCTTGAGTGCTTCA-3′(SEQ ID NO:27); F2:5′-AGTAGTGTGTGCCCGTCTGT-3′(SEQ ID NO:28)] and linker cassette specificreverse primers [R1: 5′-GACCCGGGAGATCTGAATTC-3′(SEQ ID NO:29); R2:5′-AGTGGCACAGCAGTTAGG-3′(SEQ ID NO:30)]. Between rounds of nested PCR,products were purified using streptavidin-coated magnetic beads.Products were visualized on 2% TAE agarose gels. For sequencing,products were gel purified and cloned into pCR2.1-TOPO, transformed intoE. coli Top10, selected on LB-Amp-Xgal plates, and amplified by colonyPCR using M13F/R.

Functional Assessment of Integration Sites.

Sequence products from LAM-PCR that were verified to contain proviralLTR sequence were masked for known genomic repeats and proviralfeatures. The resulting sequence was aligned to the dog genome (CanFam2.0, May 2005 assembly) using the Blat (BLAST-like alignment tool)server at UCSC. Sequences mapping to a unique location in the genome at95% similarity were selected and integration sites were determined asthe base in the genomic alignment flanking the proviral LTR sequence.For each site, the closest RefSeq gene was determined and compared to alist of human cancer orthologs.

Detection of Biologically Active Human FVIII (FVIII:C).

Lysates of 1×10⁸ platelets/ml were tested for FVIII:C using aChromogenix Coatest® SP4 FVIII kit (DiaPharma, Franklin, Ohio).12Duplicate samples of supernatant were placed in uncoated wells of a96-well microtiter plate (25 μl/well) and assay components(phospholipid, Factor IXa, Factor X, and calcium chloride) were added,and incubated for 10 min at 37° C. The chromogenic Factor Xa substrateS-675 was added, and the plate was transferred to a Wallac Victor2microplate reader preset at 37° C. The Factor Xa-dependent conversion ofS-2675 is directly related of the amount of FVIII:C in each well. Astandard curve was constructed by plotting known amounts of recombinanthuman FVIII (Kogenate; Bayer Healthcare Pharmaceuticals, Berkeley,Calif.) diluted in platelet lysate buffer using Vmax at 405 nm. The Vmaxof each reaction was converted to units of FVIII:C activity using thekinetic software program, SOFTmax, v.2.34 (Molecular Devices). The FVIIIactivity was measured by an endpoint reading at 405 nm, a backgroundreading at 490 nm was subtracted from 405 nm. The total maximumFVIII:C/dog was calculated by multiplying the mean FVIII:C U/ml/1×108platelets×92 ml blood/kg×dog weight (kg)×(2×10⁸ platelets)/1 ml blood)using measured values recorded in Table 1 and FIG. 6.

Whole Blood Clotting Time (WBCT) Assay.

WBCT is a modification of the Lee-White clotting time using twosiliconized glass tubes (Becton-Dickinson, Rutherford, N.J.) at 28° C.(Nichols T C, et al., J Thromb Haemost 2012, 10(3): 474-476). One ml ofwhole blood was drawn and 0.5 ml blood was distributed into each tube. Atimer was started. After one minute, one tube was tilted every 30 sec,the other left undisturbed. When a clot formed in the tilted tube, thesecond tube was then tilted every 30 sec until a clot formed. The timefor formation of a fully gelled clot in the second tube was recorded asthe WBCT. Blood was collected from a hemostatically normal (WBCT7.5-12.5 min) and the three experimental dogs (F20,I42,M64) before andafter G-PBC transplant if animals had not been treated with plasma forat least one month.

Inhibitor Assay to Detect Immune Response to Human FVIII.

Canine blood plasma (F20, 142 and M64) was screened for inhibitors withan activated partial thromboplastin time (aPTT) mixing assay thatdetects inhibitory antibodies to either coagulation factor VIII or IX aspreviously described (Langdell R D, et al., J Lab Clin Med 1953, 41(4):637-647; Sahud M A. Semin Thromb Hemost 2000, 26(2): 195-203; Matrai J,et al., Hepatology 2011, 53(5): 1696-1707). Briefly, test plasmas areincubated in a 1:1 mix with normal plasma for 2 h at 37° C. and then theincubated mixture is analyzed using standard aPTT reagents. Plasma fromhemophilia A dogs with known Bethesda Inhibitor (BIU) titers thatcross-react with and inhibit human FVIII (positive control) and plasmafrom dogs without inhibitors (negative control) were assayedconcurrently for comparison.

Results

Platelet-Targeted Lentiviral Vector Design and Strategy

A luciferase reporter assay revealed that fragments of the full-lengthhuman ITGA2B gene promoter permitted comparable platelet-specific genetranscription (FIG. 1A). Three different ITGA2B promoter fragments(−1218, −889 and −673) directed similar levels of luciferase activitywithin a pro-megakaryocytic cell line. In contrast, ITGA2B promoterdriven luciferase activity remained undetectable in the other blood celllineages and an epithelial cell line. Each ITGA2B promoter encodes Etsand GATA factors permitting a high level of megakaryocyte genetranscription and a repressor region that inhibits expression withinother lineages (Prandini M H, et al., Blood 1996, 88(6): 2062-2070).20As a result, two lentiviral gene transfer vectors were tested foroptimal hematopoietic stem cell transduction efficiency and the abilityto improve hemostatic function with platelet-derived BDDFVIII inhemophilia A dogs to develop a strategy for human gene therapy. Two dogsreceived an infusion of G-PBC transduced with a lentiviral vectorencoding a fragment beginning at −889 nucleotide of the human ITGA2Bpromoter shown capable of directing megakaryocyte-specific transcriptionof BDDFVIII (FIG. 1B).21 Although FVIII is absent from platelets undernormal conditions, this approach proved successful for storing viableBDDFVIII in platelet progeny derived from tissue cultured humanCD34+G-PBC,12 and lentiviral vector-transduced bone marrow transplantedinto hemophilia A mice (Shi et al., Journal of Thrombosis andHaemostasis 2007, 5(2): 352-361). One dog received an infusion of G-PBCtransduced with a novel lentiviral vector encoding the shortest fragmentof the ITGA2B promoter (−673) designed to induce megakaryocyte-specificexpression of a hybrid molecule of BDDFVIII fused to the von WillebrandFactor (VWF) propeptide signal peptide and D2 domain (SPD2) tofacilitate trafficking of BDDFVIII into the α-granule compartment (FIG.1C) (Haberichter S L, et al., Arterioscler Thromb Vasc Biol 2002, 22(6):921-926; Haberichter S L, et al., Blood 2003, 101(4): 1384-1391). VWF isa normal α-granule constituent in human platelets (albeit absent incanine platelets) (Nichols T C, et al., Blood 1993, 81(10): 2644-2651)that serves as a carrier protein of FVIII in human and canine plasma(Kaufman R J, et al. Molecular & Cellular Biology 1989, 9(3):1233-1242).

Strategy for Hematopoietic Stem Cell Gene Therapy

To design a clinically relevant protocol, canine hematopoietic stemcells were mobilized from the bone marrow into the peripheral blood withcanine cytokines (cG-CSF & cSCF) and G-PBC apheresis was performedwithout adverse incident identical to previous studies using GT dogs(Fang et al., supra). Mononuclear lymphocytes were isolated withFicoll-Paque Plus from the apheresis product and then canine CD34antigen positive (CD34+) cells were purified by immunomagnetic selection(McSweeney P A, et al., Blood 1998, 91(6): 1977-1986). Table 1summarizes the conditions for autologous transplant of three hemophiliaA dogs transfused with approximately 3×10⁶ FVIII-transducedCD34+G-PBC/kg of body weight where each target cell was transduced withapproximately 1×104 total viral particles/CD34+G-PBC without the use ofex vivo or in vivo selection for transduced cells (Columns 4,5). Anon-myeloablative pre-transplant conditioning regimen was employed tocreate a niche in the bone marrow for the newly transplanted cells toengraft (Table 1, Column 2). The intensity of the conditioning regimenis determined by the level at which the dose becomes toxic to theorgans. Earlier studies performed with normal canine models havedemonstrated that stable allogeneic mixed donor/host hematopoieticchimerism can be safely established by the administration of a sublethaldose of busulfan (a drug preferentially toxic to hematopoietic stemcells) for pre-transplant conditioning. A recent report alsodemonstrated successful use of busulfan at 10 mg/kg for hematopoieticstem cell gene transfer to correct canine leukocyte adhesion deficiency(Bauer T R, Jr., et al., Nat Med 2008, 14(1): 93-97), followed bytransient immunosuppression with mycophenolate mofetil (MMF) andcyclosporine (CSP) after major histocompatibility complex identicalmarrow transplantation (Enssle J, et al., Hum Gene Ther 2010, 21(4):397-403). However, this level of pre-transplant conditioning regimenproved inappropriate for animals with hemophilia A, since the first dog(F20) transplanted in the current study required daily supplements withcanine (c)FVIII in the form of canine plasma products and recombinantcFVIII for three months after G-PBC transplant. Epsilon-aminocaproicacid (EACA) was also infused after G-PBC transplant until human BDDFVIIIreached a significant level in platelets. EACA is an effective syntheticinhibitor of the plasmin-plasminogen system and controls subarachnoidhemorrhage, genitourinary bleeding from many causes and dental surgeryin hemophiliacs (Griffin J D, et al., Semin Thromb Hemost 1978, 5(1):27-40). For comparison, the number of serious bleeding episodes thatrequired treatment with cFVIII supplement has been recorded 1 yearbefore and 2.5 years after G-PBC transplant for each dog (Table 1,Columns 10, 11) (Niemeyer G P, et al., Experimental Hematology 2003,31(12): 1357-1362). As a result of the observation of frequent bleedingevents with F20, a milder conditioning regimen consisting of a lowerdose of busulfan was given to the next two transplant recipients (142, 5mg/kg; M64, 7 mg/kg). In conclusion, all three dogs received transientimmune suppression (Fang et al., supra) and daily supplements of cFVIIIand EACA for three months after G-PBC transplant as a standardtransplant regimen until readily detectable human BDDFVIII levels wereobserved in platelets and hemocult tests indicated the absence of GIbleeding (Fang et al., supra). Ultimately, it was observed that 142 andM64 did not require further FVIII supplements as no severe bleedingepisodes occurred for 2.5 years after transplant (Table 1, Column 11).

Biological Studies of Platelet FVIII

Immuno-confocal microscopy was performed to determine if BDDFVIII wasbeing synthesized and stored in platelets following G-PBC transplant.Shown in FIG. 2A are images of the results of microscopic analysis ofplatelets isolated from one dog (142) that received an autologoustransplant of lentiviral vector transduced G-PBC, which represents theoutcome of analysis of all three dogs (F20, 142, M64). There was apunctuate staining pattern for a specific marker of platelet α-granules,fibrinogen (Fg) (Left Panel). Human BDDFVIII was also detected in apunctuate pattern within platelets (Middle Panel). Note that BDDFVIIIstaining co-localized frequently within Fg as evident by the appearanceof a yellow staining when the left (Fg) and middle panel (BDDFVIII) wereoverlaid indicating that both proteins could be stored together withinplatelet α-granules (Right Panel) (Wilcox et al., J Thromb Haemost 2003,1(12): 2477-2489).

Immuno-electron microscopy was performed to determine if exogenousBDDFVIII was being transported specifically to platelet α-granules.Immunogold analysis was performed on ultrathin sections of plateletswith a 1° Ab to FVIII and a 2° Ab adsorbed on 10 nm gold particles (FIG.2B). The α-granules appeared normal in size and shape within plateletsof FVIII-Deficient dogs as well as FVIII transplant recipients. BDDFVIIIis absent in platelet α-granules from a FVIII-Deficient negative control(Left Panel). In contrast, BDDFVIII was detected within a-granules andcytoplasm of platelets isolated from all three dogs (F20, 142, M64).This result is consistent with observations reported for ectopicexpression of BDDFVIII within platelets of VWF(−/−) transgenic miceaffected with von Willibrand disease (Yarovoi H, et al., Blood 2005,105(12): 4674-4676). -673ITGA2B-VWFSPD2-BDDFVIII transduced plateletsfrom M64 stored the greatest level of BDDFVIII within the α-granule(Right Panel). In addition, BDDFVIII was detected rarely within membranesystems in the platelet cytoplasm indicating that the VWFSPD2 indeed hadan increased efficiency to traffic BDDFVIII directly into the α-granulecompartment.

Immunofluorescent flow cytometric analysis of platelets confirmed thatM64 stored the greatest level of FVIII per platelet because M64platelets displayed the highest mean fluorescent intensity for detectionof FVIII followed by 142 and F20 compared to FVIII-Deficient negativecontrol platelets (FIG. 3). These results indicate that the VWFSPD2targeting construct imparts an advantage for storing BDDFVIII withinplatelets. Subsequently, use of the smallest −673ITGA2B gene promoterallows the lentiviral vector to accommodate the largest therapeuticinsert (in this case, the VWFSPD2-BDDFVIII); and therefore, may be moreuseful for gene transfer rather than the −1218 or −889 ITGA2B promoters.

A Chromogenix Coatest® SP4 FVIII assay was perform to determine ifactivated platelets could secrete a biologically active form of BDDFVIII(FVIII:C) as previously shown for activated human megakaryocytes intissue culture (Wilcox et al., Thromb Haemost 2003, 1(12): 2477-2489.12In FIG. 4 platelet lysates from a FVIII-Deficient dog show that thelevel of BDDFVIII:C background activity is virtually unchanged foruntreated (Black, −Agonist) and activated platelets (White, +Agonist).In contrast, FVIII:C activity was detected readily in the lysate ofquiescent, untreated platelets from F20, 142 and M64. Furthermore,BDDFVIII:C levels were decreased in lysates of platelets stimulated by amixture of physiological agonists of platelet activation: ADP,epinephrine, and canine PAR 1,3,4 in all three experimental dogs. Insummary, dogs that received BDDFVIII-transduced G-PBC show anappreciable decrease in FVIII:C activity only after platelet activationindicating that platelets from experimental animals can be induced tosecrete FVIII within the vasculature.

Genomic Analysis of the Lentiviral Vector

The lentiviral vector WPRE element was detected by PCR of genomic DNAisolated from leukocytes collected from F20, 142, and M64 for at least2.5 years after transplant (FIG. 5A). Real time quantitative PCR(RT-qPCR) analysis of genomic DNA isolated from peripheral bloodleukocytes revealed that the transduction efficiency for each lentiviralvector was 1% (F20), 4% (142) and 2% (M64) (Table 1, Column 8). Thedetection of lentiviral vector by genomic analysis in the absence of theappearance of insertional oncogenesis is consistent with the overallgood health of all of the dogs with frequent evaluation of peripheralblood counts and peripheral blood smears documenting normal morphologyand numbers of circulating hematopoietic cells. LinearAmplification-Mediated (LAM)-PCR was also performed to determine theintegration pattern of lentiviral vector within the genome of theexperimental dogs. FIG. 5B shows that lentiviral vector was not presentwithin the genome of a FVIII-Deficient control while multiple bandsappear to be present in the genomic DNA of transplanted dogs (F20, I42and M64). A distinct insertion site was detected specifically inchromosome 4 for F20 and chromosome 35 for M64. The results demonstratethat insertion of the lentiviral vector could be detected within 142genomic DNA, although a site of insertion could not be localized to aprecise region of the current canine genome map (Sutter N B, Ostrander EA. Dog star rising: the canine genetic system. Nat Rev Genet 2004,5(12): 900-910). In summary, the results indicate that insertionalmutagenesis had not occurred when this study was concluded (≈2.5 yearsafter transplant). This is consistent with another report that foundlentiviral vectors usually insert into benign areas of the genome inanimals and humans (Biffi A, et al., Blood 2011, 117(20): 5332-5339).

Efficacy of Platelet-Targeted Gene Therapy for Hemophilia A

It was observed previously that human hematopoietic cells could serve asa primary tissue source for the synthesis of a functional form of humanBDDFVIII (FVIII:C) within tissue-cultured human megakaryocytes (Shi Q,et al., Molecular Genetics and Metabolism 2003, 79(1): 25-33.), inperipheral blood platelets isolated from mice xeno-transplanted withBDDFVIII-transduced human G-PBC,12 and in a murine model for hemophiliaA that received a transplant of BDDFVIII-transduced bone marrow (Shi Q,et al. Journal of Thrombosis and Haemostasis 2007, 5(2): 352-361). Thecurrent study (FIG. 6) shows that FVIII:C activity (≈5-15 mU/ml/108platelets) can be detected by chromogenic analysis for at least 2.5years after autologous G-PBC transplant in each dog with the highestlevels appearing approximately one year after transplant and typicallyleveling off to ≈5-10 mU/ml/10⁸ platelets (F20, 142, M6). Samples fromFVIII-Deficient dogs served as negative controls for each time point(black line).

To determine the total level FVIII:C activity present within each animalat any given time it is noted that there is ≈2×10⁸ platelets/1.0 mlblood and there is ≈92 ml blood/kg in dogs. Using values recorded inTable 1 for weight and transduction efficiency and the mean FVIII:Clevel of each dog calculated from data points shown in FIG. 6, it isestimated that there is approximately 0.230 U (F20), 1.325 U (I42) and0.676 U (M64) FVIII:C/dog stored within all of the circulatingplatelets. To put these values in perspective, the term 1 U FVIII:C/mldefines 100% FVIII activity in the reference plasma from a normal (20kg) dog; therefore, a normal (20 kg) animal has ≈800 total units ofFVIII in its plasma volume at any given time. The results in FIG. 6 showthat multiple severe bleeding episodes occurred in each animal one yearprior to G-PBC that required a transfusion with cFVIII supplements.Note, to prevent bleeding due to the gene therapy protocol, each dogreceived daily supplements of cFVIII beginning on day one of the G-PBCtransplant protocol. EACA was also administered to the transplanted dogsuntil blood was absent from their stool, which remarkably coincided withplatelet FVIII:C levels reaching ≈5 mU/ml/10⁸ platelets. F20 (Top Panel)displayed the lowest overall platelet FVIII:C levels of ≤5 mU/ml/10⁸platelets and also experienced severe intermittent bleeding episodesthroughout the experimental follow-up of 2.5 years after transplant thatrequired administration of additional supplements in the form oftransfusions of normal canine plasma or cFVIII. This result indicatesthat 5 mU/ml/108 platelet FVIII:C appears to be a threshold level oftransgene expression that must be overcome in canine hemophilia A toachieve adequate correction of the bleeding phenotype. Transplant dog142 (Middle Panel) maintained the highest steady state of FVIII:C ofapproximately 9 mU/ml/10⁸ platelets and did not experience severebleeding requiring administration of cFVIII supplements ultimatelydemonstrating correction of the hemophilia A phenotype for at least 2.5years after transplant. M64 (Bottom Panel) reached 5 mU FVIII:C/ml/10⁸platelets earlier than the other transplant dogs with the synthesis of ahybrid SPD2FVIII molecule that obtained a mean FVIII:C activity level of8 mU/ml/10⁸ platelets. This result demonstrates that the use of eitherthe −889ITGA2B gene promoter or the −673ITGA2B gene promoter coupledwith the VWFSPD2 trafficking peptide can be used effectively to targetBDDFVIII to platelets leading to correction of the canine hemophilia Aphenotype.

The time required for whole blood to clot in a test tube was measuredfor each dog using traditional version of the Lee-White whole bloodclotting time (WBCT) assay (Nichols T C, et al., J Thromb Haemost 2012,10(3): 474-476). Hemostatically normal dogs have a mean WBCT of 10.5minutes±SD 1.4 minutes. The baseline WBCT for the FVIII-Deficient dogswas 44.5 (F20), 40.5 (I42) and >60 (M64) minutes before G-PBCtransplant. After G-PBC transplant the average WBCT decreased to 39.5(F20,n=5), 38.4 (I42,n=5) and 41.9 (M64,n=4) minutes. This result showsa very modest decrease in WBCT, which could be considered well withinthe normal variation of WBCT for FVIII-Deficient dogs. Interestingly,this result supports the inability to detect FVIII:C within the plasmaof the experimental dogs (which is an essential component for success ofthe WBCT). Thus, this outcome indicates that measurement of WBCT ex vivois not a suitable assay to predict the efficacy for platelet FVIII toimprove hemostasis in vivo because (unlike plasma FVIII) the resultsindicate that platelet-derived FVIII must be secreted from activatedplatelets following stimulation with physiological platelet agonists atthe site of vascular injury to improve hemostasis within FVIII-deficientdogs as shown in FIG. 4 and FIG. 6.

To determine if the G-PBC transplant recipients developed a humoralantibody response to the newly expressed human BDDFVIII, canine bloodplasma from (F20, I42 and M64) was screened for inhibitors with anactivated partial thromboplastin time (aPTT) mixing assay which detectsinhibitory antibodies to either coagulation factor VIII or IX. Plasmafrom hemophilia A dogs with known Bethesda Inhibitor (BIU) titers thatcross-react with and inhibit human FVIII was used as a positive controland plasma from dogs without inhibitors was assayed concurrently as anegative control for comparative analysis. The results indicate thatF20, 142, and M64 did not develop inhibitors (Table 1: Column 12). Thisresult is consistent with our inability to detect the presence ofFVIII:C in the plasma. This outcome is identical with the failure ofhemophilia A mice to develop inhibitory antibodies to the human plateletBDDFVIII and our the inability to detect FVIII:C in the plasma followingtransplant of lentiviral vector-transduced murine bone marrow (Shi Q, etal., Journal of Thrombosis and Haemostasis 2007, 5(2): 352-361). Thisfurther supports targeting transgene synthesis of BDDFVIII to plateletsas a treatment for humans with pre-existing antibodies to FVIII (Shi Q,et al., J Clin Invest 2006, 116(7): 1974-1982; Kuether et al., J ThrombHaemost 2012).

TABLE 1 Conditions for Autologous Transplant of ITGA2B-BDDFVIIITransduced CD34+ G-PBC into FVIII-Deficient Dogs Pre-Tx ConditioningTotal Viral Tx CD34(+) Tx CD34(−) Busulfan Particles PBC/kg PBC/kgWeight Dog (mg/kg) Lentivirus Vector (×10{circumflex over ( )}4)/CellInfused Infused (kg) ♂ F20 10 −889 ITGA2B-BDDFVIII 0.8  4.0 ×10{circumflex over ( )}6 2.0 × 10{circumflex over ( )}8 25.20 ♂ I42 5−889 ITGA2B-BDDFVIII 1.3 1.25 × 10{circumflex over ( )}6 2.0 ×10{circumflex over ( )}8 20.00 ♂ M64 7 −673 ITGA2B-VWFSPD2- 0.7 4.58 ×10{circumflex over ( )}6 2.6 × 10{circumflex over ( )}8 22.90 BDDFVIIIPost Tx Days Post Tx Pre Tx Post Tx Post Tx Transduction Immune SeriousSerious Inhibitor Tx Age Follow-up Dog Efficency (%)* SuppressionBleeding/yr Bleeding/yr Detection Years Years ♂ F20 1.00 MMF31/CSP705.00 7.00 0/2 6.5 2.6 ♂ I42 4.00 MMF45/CSP91 5.00 0.00 0/2 4.25 2.75 ♂M64 2.00 MMF91/CSP91 3.00 0.00 0/2 1.25 2.75 *Percent (%) peripheralblood cells positive for lentiviral vector by RT-PCR *MycophenolateMofetil is MMF *Cyclosporine is CSP

All publications, patents, patent applications and accession numbersmentioned in the above specification are herein incorporated byreference in their entirety. Although the invention has been describedin connection with specific embodiments, it should be understood thatthe invention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications and variations of thedescribed compositions and methods of the invention will be apparent tothose of ordinary skill in the art and are intended to be within thescope of the following claims.

1-27. (canceled)
 28. A composition, comprising: an expression vectorcomprising: a) an expression cassette comprising a fragment of theintegrin αIIb gene (ITGA2B) promoter; a targeting factor that targetsexpression of said gene of interest to a cancer cell or a hematopoieticcell lineage producing platelets; and b) an exogenous gene of interestoperably linked to said expression cassette, wherein said exogenous geneof interest is an anti-angiogenic agent or an anti-neoplastic agent. 29.The composition of claim 28, wherein said promoter is selected from thegroup consisting of nucleotides 18-1271 of SEQ ID NO: 21, nucleotides18-939 of SEQ ID NO:22, and nucleotides 2454-3156 of SEQ ID NO:25. 30.The composition of claim 28, wherein said anti-neoplastic agent isIL-24.
 31. The composition of claim 28, wherein said targeting factor isa fragment of the human Von Willebrand Factor propeptide (VWFpp)operably linked to a D2 domain.
 32. The composition of claim 28, whereinsaid vector is a self-inactivating vector.
 33. The composition of claim32, wherein said vector is a retroviral vector.
 34. The composition ofclaim 33, wherein said retroviral vector is a lentiviral vector.
 35. Ahematopoietic stem cell or cancer stem cell comprising the compositionof claim
 28. 36. The stem of cell of claim 35, wherein said stem cell isex vivo.
 37. A method of treating cancer, comprising: a) contacting acancer stem cell or a hematopoietic stem cell with a composition ofclaim 28 to generate a modified stem cell under conditions such thatsaid exogenous gene of interest is expressed in said modified stem cell;and b) transferring said modified stem cell into an animal.
 38. Themethod of claim 37, wherein said animal is a human.
 39. The method ofclaim 37, wherein said contacting occurs ex vivo.
 40. The method ofclaim 37, wherein said stem cells are mobilized from said animal. 41.The method of claim 37, wherein said mobilizing comprises administrationof cytokines to said animal.
 42. The method of claim 37, wherein saidtransferring treats cancer in said subject.
 43. The method of claim 37,wherein said transferring prevents angiogenesis in said cancer.
 44. Themethod of claim 37, wherein said exogenous gene is expressed in plateletprogenitors of said hematopoietic stem cell.